PROJECT SUMMARY/ABSTRACT Allergic rhinitis and asthma are highly prevalent diseases characterized by allergic airway inflammation. The airway epithelium is the first line of defense against allergens. We reported that allergenic extracts stimulate oxidative DNA damage and innate immune responses in these cells. Here we show that cytosolic double strand (ds)DNA forms a complex with several proteins in the nasal mucosa of humans with allergic rhinitis and lungs of allergic mice, but not in non-sensitized humans and mice. We named this cytoplasmic DNA- interactome ?Allergosome?. The DNA-interacting proteins in the Allergosome are: interferon regulatory factor 3 (IRF3), the DNA glycosylase Nei-Like 2 (Neil2), and Cyclic GMP-AMP synthase (cGAS). Notably, these factors associate with Stimulator of Interferon Genes (STING), TANK-binding kinase-1 (TBK1), and the pro-allergic cytokine IL-33 in the Allergosome. The formation and role of the Allergosome in allergic inflammation are gaps in knowledge that we address in this proposal. The central hypothesis of this proposal is that ?Allergic humans and mice develop an Allergosome in the cytoplasm of the airway mucosa and lungs that stimulates allergic airway inflammation?. In Aim 1 we will test the hypothesis that human subjects with allergic rhinitis, but not healthy control subjects, develop an Allergosome with sequestered IL-33 in nasal mucosal cells. Cytosolic and crosslinked nuclear extracts will be prepared from the nasal punch biopsies of ragweed-allergic and healthy subjects. The cytosolic extract of the nasal biopsies will be subjected to immunoprecipitation (IP) with anti-IRF3 antibody, and analyzed for associated DNA sequences by Chip-seq, and for associated proteins in the Allergosome. In the second Aim, we will test the hypothesis that sequestration of Neil2 in the Allergosome reduces its ability to protect promoter sites in the genome from binding NF?B and stimulating allergic airway inflammation. Cytosolic and nuclear extracts will be prepared as in Aim 1. The cytosolic extracts will be subjected to IP with anti-Neil2 antibody, and analyzed as in Aim 1. The nuclear extracts will be subjected to ChIP-Seq of the IP?d DNA with anti-Neil2 and anti-NF?B antibodies to determine their promoter occupancy, and gel shift analysis to detect NF?B binding. Recombinant WT and catalytically inactive Neil2 will be delivered to the airways of sensitized WT mice by engineered peptide carriers, and allergic inflammation will be reassessed. In Aim 3, we will test the hypothesis that STING and cGAS stabilize the Allergosome and stimulate allergic airway inflammation. Allergosome formation and CDE-induced allergic inflammation in WT mice will be compared to that of StingKO mice, lung epithelium-specific inducible cGasKO mice, and WT mice treated with pharmacologic cGAS inhibitors. These studies will define, for the first time, the central role of the Allergosome in allergic airway inflammation. We will provide proof of principle that targeting components of the Allergosome reduces allergic inflammation. In future studies, these strategies may develop into therapies that mitigate allergic inflammation in allergic rhinitis and asthma.